Project Summary Despite the clinical success of anti-estrogen therapies for the treatment of patients with estrogen receptor- positive (ER+) breast cancer, recurrences occur in ~1/3 of patients treated in the adjuvant setting and almost all patients treated in the metastatic setting. The phosphatidylinositol 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway has been implicated in anti-estrogen resistance, and drugs targeting these pathways are approved or in clinical trials. Unfortunately, nearly all ER+ breast cancers progress on these therapies as well. The high prevalence of disease recurrence in patients, despite dramatic treatment efficacy in preclinical models, led us to postulate that components of the tumor microenvironment significantly contribute to resistance to anti-estrogens and PI3K/mTOR inhibitors in ER+ breast cancer. Using a novel, microenvironment-focused approach combining high-throughput screening and bioinformatics, we uncovered fibroblast growth factor 2 (FGF2) as a potent mediator of resistance to both anti-estrogens and PI3K/mTOR inhibitors that is highly expressed in normal tissues relevant to the microenvironments of ER+ breast cancer. FGF2 rescues cells from treatment-induced apoptosis and cell cycle arrest, and rescue is abrogated by an FGF2-specific antibody or a kinase inhibitor targeting all four FGF receptors (FGFRs). We hypothesize that FGF2 mediates resistance to both anti-estrogens and PI3K/mTOR inhibitors, alone and in combination, through pathways downstream of FGFRs that converge on signaling nodes that modulate cell fate, and that resistance can be abrogated with FGF2-targeted therapeutics in settings of both primary and metastatic ER+ breast cancer. Specific Aim 1 will determine the precise mechanism(s) of FGF2-mediated rescue from anti- estrogens and PI3K/mTOR inhibitors, which may provide novel tumor-specific therapeutic targets necessary for the resistance phenotype. FGFR isoform specificity will be determined using genetic approaches, and identification of downstream signaling networks involved in FGF2-mediated resistance will be achieved using immunoblotting and phosphoproteomics. Specific Aim 2 will use 3 microenvironmentally-relevant tumor models of ER+ breast cancer to assess whether targeting FGF2 enhances response to anti-estrogens and PI3K inhibitors. FGF2 is highly expressed in mammary tissue, bone, and primary fibroblasts, so we will utilize models of these tumor microenvironments: 1) an orthotopic primary breast cancer patient-derived xenograft (PDX) model, 2) a bone metastasis cell line-derived xenograft model developed by the applicant, and 3) a murine ER+ mammary adenocarcinoma model that heavily recruits host-derived FGF2-secreting fibroblasts. Through these studies, we will uncover potentially druggable protein targets that are required for FGF2- mediated drug resistance, and define the appropriate clinical setting for FGF2-directed therapy for ER+ breast cancer. These advances will improve therapeutic strategies to abrogate and prevent resistance to anti- estrogens and PI3K/mTOR-directed therapies in patients with ER+ breast cancer.